摘要
本文利用定点突变技术改造微小毛霉凝乳酶，将突变体（E13D）在毕赤酵母中表达，并对其进行纯化及性质研究，此突变酶水解活性有明显降低。通过SDS—PAGE电泳鉴定纯化后酶的分子质量为46kDa。当pH5．0～7．0，温度为60℃时酶活力最高，且pH从8．0到5．0范围内，酶活力随着pH降低而逐渐升高。Fe^2＋，Fe^3＋，Cu^＋和Zn^2＋是酶的机制剂，同时凝乳活力受1．10-Phenantrolin和peps／atinA抑制。Ca^2＋，Mn^2＋和Mg^2＋是酶的激活剂，对酶活力有一定的催化作用。利用该突变酶制作干酪，进行质构分析，同时与未突变酶及商品凝乳酶制作的干酪进行比对分析，发现除突变酶制作的干酪的可溶性氮含量低、硬度较其它两种干酪高外，其它性质没有明显的变化，这一结果某种程度上说明突变降低了酶的水解活性，使其在干酪成熟过程中抑制了干酪的过度水解，具有替代牛凝乳酶的潜力。
Site-directed mutagenesis was carried out to alter properties of Mucor pusillus rennet （MPR） for a substitution of commercial chymosin. One mutant, E13D, with decreased proteolytic activity was expressed in Pichia pastoris, purified and characterized. The molecular weight of the puried enzyme was estimated to be 46 kDa by SDS-PAGE. The maximum enzyme activity was at a wide range of pHs （5.0-7.0） and 60℃. The milk-clotting activity （MCA） increased with a decrease in the pH from 8.0 to 5.0. The enzyme was inhibited by heavy metal ions （Fe^2＋, Fe^3＋, Cu^＋ and Zn=^2＋）, 1.10-Phenantrolin and pepstatin A. Reactivation of the enzyme activity with Ca^2＋, Mn^2＋ and Mg^2＋ indicated the importance of these metals in the catalytic function of the enzyme. Further texture analysis of cheddar cheeses made by non-mutant rennet, mutant E13D rennet and commercial rennet found that there was no significant difference between mutant rennet cheese and commercial coagulant cheese, except for lower soluble nitrogen content and higher value of hardness of mutant cheese than those of the non-mutant. The result implicated that, in some extent, the mutant rennet could decrease hydrolysis of protein during ripening of cheese, probably to be candidate for a useful milk coatulant.
